Bone destruction by osteoclasts is essential for normal bone development. Osteoclast deficiency leads to osteopetrosis, which is characterized by increased bone mass and may lead to bone deformities or in severe cases, to death. Increased numbers and activity of osteoclasts, on the other hand, cause increased bone resorption, and may lead to osteoporosis and other osteolytic diseases. A better understanding of the molecular regulation of osteoclast formation, activity, and survival will provide novel targets for therapeutic intervention in the control of these diseases. Recently, we identified a gene, nha-oc/NHA2, which is strongly up-regulated during RANKL-induced osteoclast differentiation in vitro and in vivo. nha-oc/NHA2 encodes a novel cation/proton antiporter (CPA) that is selectively expressed in osteoclasts. NHA-oc/NHA2 protein localizes to the mitochondria, where it mediates Na+dependent mitochondrial swelling. NHA-oc/NHA2 is therefore the first characterized mammalian mitochondrial CPA2. RNA silencing of nha-oc/nha2 reduces osteoclast differentiation and bone resorption, strongly indicating a role for NHA-oc/NHA2 in these processes. In Aim 1, we will characterize the role of NHA-oc/NHA2 in bone homeostasis in vivo by using a knockout mouse model carrying a mutagenic retroviral insertion in the nha-oc/NHA2 locus. In Aim 2, we will characterize the role of NHA-oc/NHA2 on pathological bone loss in vivo. For that, we will compare bone loss induced by ovariectomy in wild type and homozygous mutant mice. The effects of nha-oc/NHA2 on osteoclast formation and bone resorption in vivo will be determined using a variety of analytic approaches for bone. Our goal is to establish the role that NHA-oc/NHA2 plays in osteoclast differentiation and function, and ultimately in regulating bone mass in vivo. These studies may identify a new candidate gene involved in the development of human osteopetrosis as well as provide an important new target for anti-resorptive therapies. PUBLIC HEALTH RELEVANCE: Osteoclasts are cells that are responsible for bone removal ('resorption') during normal bone development and maintenance. In contrast, abnormal osteoclast numbers and/or activity cause a spectrum of diseases ranging from osteopetrosis to osteoporosis. This project seeks to determine the role of a novel gene that we have discovered in osteoclasts, termed 'NHA-oc/NHA2', in osteoclast formation, function and ultimately in regulating bone mass in vivo. This work will aid us in the design of appropriate new therapies based on drugs that interfere with NHA-oc/NHA2 activity for the prevention of pathological bone loss.